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Ovule initiation is a key step that strongly influences ovule number and seed yield. Notably, mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signaling produce more ovules and have a higher seed number per silique (SNS) than wild-type plants. Here, we crossed BR- and CK-related mutants to test whether these phytohormones function together in ovule initiation. We determined that simultaneously enhancing BR and CK contents led to higher ovule and seed numbers than enhancing BR or CK separately, and BR and CK enhanced each other. Further, the BR-response transcription factor BZR1 directly interacted with the CK-response transcription factor ARABIDOPSIS RESPONSE REGULATOR1 (ARR1). Treatments with BR or BR plus CK strengthened this interaction and subsequent ARR1 targeting and induction of downstream genes to promote ovule initiation. Enhanced CK signaling partially rescued the reduced SNS phenotype of BR-deficient/insensitive mutants whereas enhanced BR signaling failed to rescue the low SNS of CK-deficient mutants, suggesting that BR regulates ovule initiation and SNS through CK-mediated and CK-independent pathways. Our study thus reveals that interaction between BR and CK promotes ovule initiation and increases seed number, providing important clues for increasing the seed yield of dicot crops. This article is protected by copyright. All rights reserved.

RNA silencing is an evolutionarily homology-based gene inactivation mechanism and plays critical roles in plant immune responses to acute or chronic virus infections, which often pose serious threats to agricultural productions. Plant antiviral immunity is triggered by virus-derived small interfering RNAs (vsiRNAs) and functions to suppress virus further replication via a sequence-specific degradation manner. Through plant-virus arms races, many viruses have evolved specific protein(s), known as viral suppressors of RNA silencing (VSRs), to combat plant antiviral responses. Numerous reports have shown that VSRs can efficiently curb plant antiviral defense response via interaction with specific component(s) involved in the plant RNA silencing machinery. Members in the family Closteroviridae (closterovirids) are also known to encode VSRs to ensure their infections in plants. In this review, we will focus on the plant antiviral RNA silencing strategies, and the most recent developments on the multifunctional VSRs encoded by closterovirids. Additionally, we will highlight the molecular characters of phylogenetically-associated closterovirids, the interactions of these viruses with their host plants and transmission vectors, and epidemiology.

Stemonae Radix (dried roots of Stemona japonica (Bl.) Miq., Stemona sessilifolia (Miq.) Miq., and Stemona tuberosa Lour.), a widely used traditional Chinese medicine (TCM), is also known as “Baibu” (Chinese: 百部). Stemonae Radix has a wide range of pharmacological effects, mainly used to treat respiratory diseases and as an insecticide. In this review, with the help of both published materials (such as ancient and modern books, monographs on medicinal plants, and the pharmacopoeia of different countries) and electronic databases (such as PubMed, Web of Science, and Google Scholar), we have summarized the up-to-date research progress in botany, traditional application, phytochemistry, pharmacology, toxicity, and quality control, and discussed the research prospects and development direction of Stemonae Radix and its origin plants. Up to now, more than 170 compounds were isolated and identified from the three official species of Stemonae Radix, mainly including Stemona alkaloids, Stemona stilbenoids and other components. This medicine has also exhibited pharmacological activities, including antitussive, antiviral, insecticidal, antitumor, antioxidant, and therapeutic effects on lung diseases. However, pharmacological reports most often attribute activities to various extracts, rather than specific compounds. This hampers the establishment of a connection between chemical constituents and pharmacological activities, resulting in a difficulty to identify the major active compounds. Therefore, in the future, it is necessary to design experiments to deeply understand the development and clinical use of Stemonae Radix.

Positive-stranded RNA viruses usually remodel host endomembrane system to form virus-induced intracellular vesicles for replication during infections. The genus Potyvirus of Potyviridae represents the largest number of positive single-stranded RNA viruses and causes great damage on crop production worldwide. Though potyviruses have wide host ranges, each potyvirus infects relatively limited host species. Phylogenesis and host range analysis can divide potyviruses into monocot-infecting and dicot-infecting groups, suggesting that some infection mechanism, probably on replication may be distinct for each group. Comprehensive studies on the model dicot-infecting turnip mosaic virus indicated that the 6K2-induced replication vesicles are derived from endoplasmic reticulum (ER) and subsequently target chloroplasts for viral genome replication. However, we have no knowledge on the replication site of monocot-infecting potyviruses. In this study, we firstly show that the precursor 6K2-VPg-Pro polyproteins of dicot-infecting potyviruses and monocot-infecting potyviruses phylogenetically cluster in two separate groups. With a typical gramineae-infecting potyvirus sugarcane mosaic virus (SCMV), we found that SCMV replicative double-stranded RNA (dsRNA) forms aggregates in cytoplasm but does not associate with chloroplasts. SCMV 6K2-VPg-Pro-induced vesicles colocalize with replicative dsRNA. Moreover, SCMV 6K2-VPg-Pro-induced structures target multiple intracellular organelles including ER, Golgi apparatus, mitochondria and peroxisomes, and have no evident association with chloroplasts. In conclusion, SCMV remodels multiple intracellular organelles for its genomic RNA replication.

Pennisetum plants (Pennisetum alopecuroides L.), displaying a dwarfing phenotype along with delayed flowering and mosaic symptom on leaves, were found in Beijing, China. Flexuous filamentous particles with a size of approximate 15 × 850 nm were observed in symptomatic leaves via transmission electron microscopy. Deep sequencing of small RNAs (sRNA) from symptomatic leaves and analysis of sRNA populations were then conducted to determine the genome sequence of the viral agent in diseased plant tissues. It showed that the viral agent had one positive-sense and single-stranded RNA genome, which consisted of 9717 nucleotides (nts) excluding poly(A) tail. The complete viral genome contained a large open reading frame, encoding a polyprotein of 3131 amino acids (aa). Sequence comparison and phylogenetic analysis demonstrated that the viral agent belonged to the genus Potyvirus in the family Potyviridae. In the cladogram it was most closely related to johnsongrass mosaic virus, sharing 72% nt and 65% aa sequence identity. This viral agent was provisionally named pennisetum alopecuroides mosaic virus (PalMV). Subsequently, it was confirmed that PalMV is the causal agent of this new disease in P. alopecuroides by Koch’s postulates and reverse transcription-polymerase chain reaction analysis. Moreover, maize, millet, wheat, sorghum and rice plants were experimentally infected by PalMV via rub inoculation. Consequently, we proposed that PalMV could be a potentially dangerous virus threating a wide range of cereal crops.

Background Ginsenoside Rh2 (GRh2) is a characterized component in red ginseng widely used in Korea and China. GRh2 exhibits a wide range of pharmacological activities, such as anti-inflammatory, antioxidant, and anticancer properties. However, its effects on Toxoplasma gondii (T. gondii) infection have not been clarified yet. Methods The effect of GRh2 against T. gondii was assessed under in vitro and in vivo experiments. The BV2 cells were infected with tachyzoites of T. gondii RH strain, and the effects of GRh2 were evaluated by MTT assay, morphological observations, immunofluorescence staining, a trypan blue exclusion assay, reverse transcription PCR, and Western blot analyses. The in vivo experiment was conducted with BALB/c mice inoculated with lethal amounts of tachyzoites with or without GRh2 treatment. Results and conclusion The GRh2 treatment significantly inhibited the proliferation of T. gondii under in vitro and in vivo studies. Furthermore, GRh2 blocked the activation of microglia and specifically decreased the release of inflammatory mediators in response to T. gondii infection through TLR4/NF-κB signaling pathway. In mice, GRh2 conferred modest protection from a lethal dose of T. gondii. After the treatment, the proliferation of tachyzoites in the peritoneal cavity of infected mice markedly decreased. Moreover, GRh2 also significantly decreased the T. gondii burden in mouse brain tissues. These findings indicate that GRh2 exhibits an anti–T. gondii effect and inhibits the microglial activation through TLR4/NF-κB signaling pathway, providing the basic pharmacological basis for the development of new drugs to treat toxoplasmic encephalitis.

Purpose Evidence indicates that the actin-binding protein Coronin 3, which is aberrantly expressed in various cancers, is associated with cancer development and progression. However, little is known about the role of Coronin 3 in glioma tumorigenesis. Here, we aimed to explore the biological function and regulatory mechanism of Coronin 3 in glioblastoma (GBM). Materials and methods Coronin 3 level in human GBM clinical samples and cell lines was investigated. The shRNA knockdown strategy was used to assess the tumor characteristics of GBM cell lines. The role of β-catenin in Coronin 3-mediated oncogenic phenotypes was evaluated. Results Coronin 3 was found to be highly upregulated in glioma cell lines. Furthermore, knockdown of Coronin 3 significantly inhibited the growth of glioma cells both in vivo and in vitro and suppressed the expression of Wnt/β-catenin pathway genes, including β-catenin, Cyclin D1, and c-Myc. Moreover, we demonstrated that Coronin 3 regulates the expression of β-catenin in glioma. Our results revealed that Coronin 3-stimulated tumor growth was β-catenin-dependent. Conclusion Our study reveals a new molecular mechanism of Coronin 3 in promoting glioma growth and development through regulating the Wnt/β-catenin signaling pathway.

Two types of tonoplast proton pumps, H+ -pyrophosphatase (V-PPase) and the H+ -ATPase (V-ATPase), establish the proton gradient that powers molecular traffic across the tonoplast thereby facilitating turgor regulation and nutrient homeostasis. However, how proton pumps regulate development remains unclear. In this study, we investigated the function of two types of proton pumps in Arabidopsis embryo development and pattern formation. While disruption of either V-PPase or V-ATPase had no obvious effect on plant embryo development, knocking out both resulted in severe defects in embryo pattern formation from the early stage. While the first division in wild-type zygote was asymmetrical, a nearly symmetrical division occurred in the mutant, followed by abnormal pattern formation at all stages of embryo development. The embryonic defects were accompanied by dramatic differences in vacuole morphology and distribution, as well as disturbed localisation of PIN1. The development of mutant cotyledons and root, and the auxin response of mutant seedlings supported the hypothesis that mutants lacking tonoplast proton pumps were defective in auxin transport and distribution. Taking together, we proposed that two tonoplast proton pumps are required for vacuole morphology and PIN1 localisation, thereby controlling vacuole and auxin-related developmental processes in Arabidopsis embryos and seedlings.

Vacuoles are organelles in plant cells that play pivotal roles in growth and developmental regulation. The main functions of vacuoles include maintaining cell acidity and turgor pressure, regulating the storage and transport of substances, controlling the transport and localization of key proteins through the endocytic and lysosomal-vacuolar transport pathways, and responding to biotic and abiotic stresses. Further, proteins localized either in the tonoplast (vacuolar membrane) or inside the vacuole lumen are critical for fruit quality. In this review, we summarize and discuss some of the emerging functions and regulatory mechanisms associated with plant vacuoles, including vacuole biogenesis, vacuole functions in plant growth and development, fruit quality, and plant-microbe interaction, as well as some innovative research technology that has driven advances in the field. Together, the functions of plant vacuoles are important for plant growth and fruit quality. The investigation of vacuole functions in plants is of great scientific significance and has potential applications in agriculture.

Gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19), a novel IFN-β/retinoic acid-inducible gene product, has been identified as a potential tumor suppressor, which is associated with the inhibition of tumor growth. GRIM-19 has been demonstrated to be downregulated in the ovarian tissue of patients with breast cancer, however, its role in breast cancer remains to be fully elucidated. In the present study, a recombinant eukaryotic expression plasmid carrying GRIM-19 was constructed and then transfected into the MCF7 human breast cancer cell line to examine its effects on breast cancer cell growth, migration and invasion using several in vitro approaches. The results demonstrated that upregulation GRIM-19 in the MCF7 cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis. Additionally, upregulation of GRIM-19 also suppressed the secretion of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF). It was also demonstrated that the activation of signal transducer and activator of transcription 3 (STAT3) was downregulated by the expression of GRIM-19. These results revealed that overexpression of the GRIM-19 gene may be an effective approach to control the growth and invasion of human breast cancer cells.

Summary Sugarcane mosaic virus (SCMV) is a pathogen of worldwide importance that causes dwarf mosaic disease on maize (Zea mays). Until now, few maize genes/proteins have been shown to be involved in resistance to SCMV. In this study, we characterized the role of maize phenylalanine ammonia‐lyases (ZmPALs) in accumulation of the defence signal salicylic acid (SA) and in resistance to virus infection. SCMV infection significantly increased SA accumulation and expression of SA‐responsive pathogenesis‐related protein genes (PRs). Interestingly, exogenous SA treatment decreased SCMV accumulation and enhanced resistance. Both reverse transcription‐coupled quantitative PCR and RNA‐Seq data confirmed that expression levels of at least four ZmPAL genes were significantly up‐regulated upon SCMV infection. Knockdown of ZmPAL expression led to enhanced SCMV infection symptom severity and virus multiplication, and simultaneously resulted in decreased SA accumulation and PR gene expression. Intriguingly, application of exogenous SA to SCMV‐infected ZmPAL‐silenced maize plants decreased SCMV accumulation, showing that ZmPALs are required for SA‐mediated resistance to SCMV infection. In addition, lignin measurements and metabolomic analysis showed that ZmPALs are also involved in SCMV‐induced lignin accumulation and synthesis of other secondary metabolites via the phenylpropanoid pathway. In summary, our results indicate that ZmPALs are required for SA accumulation in maize and are involved in resistance to virus infection by limiting virus accumulation and moderating symptom severity.

Background and Purpose— Inhibition of brain NKCC1 (Na + -K + -Cl − cotransporter 1) with bumetanide (BMT) is of interest in ischemic stroke therapy. However, its poor brain penetration limits the application. In this study, we investigated the efficacy of 2 novel NKCC1 inhibitors, a lipophilic BMT prodrug STS5 (2-(Dimethylamino)ethyl 3-(butylamino)-4-phenoxy-5-sulfamoyl-benzoate;hydrochloride) and a novel NKCC1 inhibitor STS66 (3-(Butylamino)-2-phenoxy-5-[(2,2,2-trifluoroethylamino)methyl]benzenesulfonamide), on reducing ischemic brain injury. Methods— Large-vessel transient ischemic stroke in normotensive C57BL/6J mice was induced with 50-min occlusion of the middle cerebral artery and reperfusion. Focal, permanent ischemic stroke in angiotensin II (Ang II)–induced hypertensive C57BL/6J mice was induced by permanent occlusion of distal branches of middle cerebral artery. A total of 206 mice were randomly assigned to receive vehicle DMSO, BMT, STS5, or STS66. Results— Poststroke BMT, STS5, or STS66 treatment significantly decreased infarct volume and cerebral swelling by ≈40% to 50% in normotensive mice after transient middle cerebral artery occlusion, but STS66-treated mice displayed better survival and sensorimotor functional recovery. STS5 treatment increased the mortality. Ang II–induced hypertensive mice exhibited increased phosphorylatory activation of SPAK (Ste20-related proline alanine-rich kinase) and NKCC1, as well as worsened infarct and neurological deficit after permanent distal middle cerebral artery occlusion. Conclusions— The novel NKCC1 inhibitor STS66 is superior to BMT and STS5 in reducing ischemic infarction, swelling, and neurological deficits in large-vessel transient ischemic stroke, as well as in permanent focal ischemic stroke with hypertension comorbidity.

Primary dysmenorrhea is a common disease among females in their reproductive age and adolescents. The main complaint is lower abdominal pain during menstruation. Females, who suffer from dysmenorrhea, widely use nonsteroidal anti-inflammatory drugs that reduce muscle spasm by inhibiting prostaglandin synthesis and vasopressin secretion. However, there are side effects when patients take them for a long time. It is therefore against this backdrop that herbal medicines are suggested as an alternative source of treatment for primary dysmenorrhea. In this paper, a review of studies demonstrating the relieving of uterine contraction and reduction in prostaglandin synthesis by alternative sources such as traditional Chinese medicines (TCM) is spelt out. TCM conceptualizes that, menstrual pain resulting from qi is due to stagnated and retained blood. Blood deficiency and coldness in the whole human body are two additional causes of dysmenorrhea. Therefore, based on these, the main focus of treatment is directed at relieving the above symptoms. Chinese herbal prescriptions exert their effects through these mechanisms: prostaglandin reduction, inhibition of cyclooxygenase, intracellular Ca2+, and nitric oxide, declining malondialdehyde, and reverse increasing superoxide dismutase. Hence, Chinese herbal prescriptions, present perhaps a more beneficial efficacious alternative in the treatment of primary dysmenorrhea, more especially in the confines of complementary and alternative medicine.

Pathogens disturb alternative splicing patterns of infected eukaryotic hosts. However, in plants it is unknown if this is incidental to infection or represents a pathogen-induced remodeling of host gene expression needed to support infection. Here, we compared changes in transcription and protein accumulation with changes in transcript splicing patterns in maize (Zea mays) infected with the globally important pathogen sugarcane mosaic virus (SCMV). Our results suggested that changes in alternative splicing play a major role in determining virus-induced proteomic changes. Focusing on maize phytoene synthase1 (ZmPSY1), which encodes the key regulatory enzyme in carotenoid biosynthesis, we found that although SCMV infection decreases total ZmPSY1 transcript accumulation, the proportion of splice variant T001 increases by later infection stages so that ZmPSY1 protein levels are maintained. We determined that ZmPSY1 has two leaf-specific transcripts, T001 and T003, distinguished by differences between the respective 3'-untranslated regions (UTRs). The shorter 3'-UTR of T001 makes it the more efficient mRNA. Nonsense ZmPSY1 mutants or virus-induced silencing of ZmPSY1 expression suppressed SCMV accumulation, attenuated symptoms, and decreased chloroplast damage. Thus, ZmPSY1 acts as a proviral host factor that is required for virus accumulation and pathogenesis. Taken together, our findings reveal that SCMV infection-modulated alternative splicing ensures that ZmPSY1 synthesis is sustained during infection, which supports efficient virus infection.

Fungal infections have emerged as a major global threat to human health because of the increasing incidence and mortality rates every year. The emergence of drug resistance and limited arsenal of antifungal agents further aggravates the current situation resulting in a growing challenge in medical mycology. Here, we identified that ponatinib, an FDA-approved antitumour drug, significantly enhanced the activity of the azole fluconazole, the most widely used antifungal drug. Further detailed investigation of ponatinib revealed that its combination with fluconazole displayed broad-spectrum synergistic interactions against a variety of human fungal pathogens such as Candida albicans, Saccharomyces cerevisiae and Cryptococcus neoformans. Mechanistic insights into the mode of action unravelled that ponatinib reduced the efflux of fluconazole via Pdr5 and suppressed the expression of the proton pump, Pma1. Taken together, our study identifies ponatinib as a novel antifungal that enhances drug activity of fluconazole against diverse fungal pathogens.

Plant ovule initiation determines the maximum of ovule number and has a great impact on the seed number per fruit. The detailed processes of ovule initiation have not been accurately described, although two connected processes, gynoecium and ovule development, have been investigated. Here, we report that ovules initiate asynchronously. The first group of ovule primordia grows out, the placenta elongates, the boundaries of existing ovules enlarge and a new group of primordia initiates from the boundaries. The expression pattern of different marker genes during ovule development illustrates that this asynchronicity continues throughout whole ovule development. PIN-FORMED1 polar distribution and auxin response maxima correlate with ovule primordia asynchronous initiation. We have established computational modeling to show how auxin dynamics influence ovule primordia initiation. Brassinosteroid signaling positively regulates ovule number by promoting placentae size and ovule primordia initiation through strengthening auxin response. Transcriptomic analysis demonstrates numerous known regulators of ovule development and hormone signaling, and many new genes are identified that are involved in ovule development. Taken together, our results illustrate that the ovule primordia initiate asynchronously and the hormone signals are involved in the asynchrony., Summary: Ovule primordia initiation, which determines the maximum ovule number and subsequent seed number in Arabidopsis, is asynchronous and is regulated by PIN1 polar distribution and the auxin response.

Disturbed blood flow has been recognized to promote platelet aggregation and thrombosis via increasing accumulation of von Willebrand factor (VWF) at the arterial post-stenotic sites. The mechanism underlying the disturbed-flow regulated endothelial VWF production remains elusive. Here we described a mouse model, in which the left external carotid artery (LECA) is ligated to generate disturbed flow in the common carotid artery. Ligation of LECA increased VWF accumulation in the plasma. Carotid arterial thrombosis was induced by ferric chloride (FeCl3) application and the time to occlusion in the ligated vessels was reduced in comparison with the unligated vessels. In vitro, endothelial cells were subjected to oscillatory shear (OS, 0.5 ± 4 dynes/cm2) or pulsatile shear (PS, 12 ± 4 dynes/cm2). OS promoted VWF secretion as well as the cell conditioned media-induced platelet aggregation by regulating the intracellular localization of vesicle-associated membrane protein 3 (VAMP3) and synaptosomal-associated protein 23 (SNAP23). Disruption of vimentin intermediate filaments abolished the OS-induced translocation of SNAP23 to the cell membrane. Knockdown of VAMP3 and SNAP23 reduced the endothelial secretion of VWF. Systemic inhibition of VAMP3 and SNAP23 by treatment of mice with rapamycin significantly ameliorated the FeCl3-induced thrombogenesis, whereas intraluminal overexpression of VAMP3 and SNAP23 aggravated it. Our findings demonstrate VAMP3 and SNAP23 as potential targets for preventing the disturbed flow-accelerated thrombus formation.

Epilepsy is a common brain disorder, repeated seizures of epilepsy may lead to a series of brain pathological changes such as neuronal or glial damage. However, whether circular RNAs are involved in neuronal injury during epilepsy is not fully understood. Here, we screened circIgf1r in the status epilepticus model through circRNA sequencing, and found that it was upregulated after the status epilepticus model through QPCR analysis. Astrocytes polarizing toward neurotoxic A1 phenotype and neurons loss were observed after status epilepticus. Through injecting circIgf1r siRNA into the lateral ventricle, it was found that knocking down circIgf1r in vivo would induce the polarization of astrocytes to phenotype A2 and reduce neuronal loss. The results in vitro further confirmed that inhibiting the expression of circIgf1r in astrocytes could protect neurons by converting reactive astrocytes from A1 to the protective A2. In addition, knocking down circIgf1r in astrocytes could functionally promote astrocyte autophagy and relieve the destruction of 4-AP-induced autophagy flux. In terms of mechanism, circIgf1r promoted the polarization of astrocytes to phenotype A1 by inhibiting autophagy. Taken together, our results reveal circIgf1r may serve as a potential target for the prevention and treatment of neuron damage after epilepsy.

Although Pseudoalteromonas is an abundant, ubiquitous, marine algae-associated bacterial genus, there is still little information on their phages. In the present study, a marine phage AL, infecting Pseudoalteromonas marina, was isolated from the coastal waters off Qingdao. The AL phage is a siphovirus with an icosahedral head of 53 ± 1 nm and a non-contractile tail, length of 99 ± 1 nm. A one-step growth curve showed that the latent period was approximately 70 min, the rise period was 50 min, and the burst size was 227 pfu/cell. The genome sequence of this phage is a 33,582 bp double-stranded DNA molecule with a GC content of 40.1 %, encoding 52 open reading frames (ORFs). The order of the functional genes, especially those related to the structure module, is highly conserved and basically follows the common pattern used by siphovirus. The stable order has been formed during the long-term evolution of phages in the siphovirus group, which has helped the phages to maintain their normal morphology and function. Phylogenetic trees based on the major capsid protein (mcp) and genome-wide sequence have shown that the AL phage is closely related to four Pseudoalteromonas phages, including PHS21, PHS3, SL25 and Pq0. Further analysis using all-to-all BLASTP also confirmed that this phage shared high sequence homology with the same four Pseudoalteromonas phages, with amino acid sequence identities ranging from 44 % to 71 %. In particular, their similarity in virion structure module may imply that these phages share common assembly mechanism characteristics and infection pathways. Pseudoalteromonas phage AL not only provides basic information for the further study of the evolution of Pseudoalteromonas phages and interactions between marine phage and host but also helps to explain the unknown viral sequences in the metagenomic databases.

The abundant and widespread unicellular cyanobacteria Synechococcus plays an important role in contributing to global phytoplankton primary production. In the present study, two novel cyanomyoviruses, S-N03 and S-H34 that infected Synechococcus MW02, were isolated from the coastal waters of the Yellow Sea. S-N03 contained a 167,069-bp genome comprising double-stranded DNA with a G + C content of 50.1%, 247 potential open reading frames and 1 tRNA, S-H34 contained a 167,040-bp genome with a G + C content of 50.1%, 246 potential open reading frames and 5 tRNAs. These two cyanophages contain fewer auxiliary metabolic genes (AMGs) than other previously isolated cyanophages. S-H34 in particular, is currently the only known cyanomyovirus that does not contain any AMGs related to photosynthesis. The absence of such common AMGs in S-N03 and S-H34，their distinct evolutionary history and ecological features imply that the energy for phage production might be obtained from other sources rather than being strictly dependent on the maintenance of photochemical ATP under high light. Phylogenetic analysis showed that the two isolated cyanophages clustered together and had a close relationship with two other cyanophages of low AMG content. Comparative genomic analysis, habitats and hosts across 81 representative cyanomyovirus showed that cyanomyovirus with less AMGs content all belonged to Synechococcus phages isolated from eutrophic waters. The relatively small genome size and high G + C content may also relate to the lower AMG content, as suggested by the significant correlation between the number of AMGs and G + C%. Therefore, the lower content of AMG in S-N03 and S-H34 might be a result of viral evolution that was likely shaped by habitat, host, and their genomic context. The genomic content of AMGs in cyanophages may have adaptive significance and provide clues to their evolution.

Due to the increasing prevalence of pathogenic fungal infections, the emergence of antifungal resistant clinical isolates worldwide, and the limited arsenal of available antifungals, developing new antifungal strategies is imperative. In this study, we screened a library of 1068 FDA-approved drugs to identify hits that exhibit broad-spectrum antifungal activity. Robenidine, an anticoccidial agent which has been widely used to treat coccidian infections of poultry and rabbits, was identified in this screen. Physiological concentration of robenidine (8 μM) was able to significantly inhibit yeast cell growth, filamentation and biofilm formation of Candida albicans – the most extensively studied human fungal pathogen. Moreover, we observed a broad-spectrum antifungal activity of this compound against fluconazole resistant clinical isolates of C. albicans, as well as a wide range of other clinically relevant fungal pathogens. Intriguingly, robenidine-treated C. albicans cells were hypersensitive to diverse cell wall stressors, and analysis of the cell wall structure by transmission electron microscopy (TEM) showed that the cell wall was severely damaged by robenidine, implying that this compound may target the cell wall integrity signaling pathway. Indeed, upon robenidine treatment, we found a dose dependent increase in the phosphorylation of the cell wall integrity marker Mkc1, which was decreased after prolonged exposure. Finally, we provide evidence by RNA-seq and qPCR that Rlm1, the downstream transcription factor of Mkc1, may represent a potential target of robenidine. Therefore, our data suggest that robenidine, a FDA approved anti-coccidiosis drug, displays a promising and broadly effective antifungal strategy, and represents a potentially repositionable candidate for the treatment of fungal infections.

Synechococcus dominate picocyanobacterial communities in coastal environments. However, only a few Synechococcus phages have been described from the coastal seas of the Northwest Pacific Ocean. Here a new Synechococcus phage, S-B43 was isolated from the Bohai Sea, a semi-closed coastal sea of the Northwest Pacific Ocean. S-B43 is a member of Myoviridae, containing 275 predicted open reading frames. Fourteen auxiliary metabolic genes (AMG) were identified from the genome of S-B43, including five photosynthetic associated genes and several AMGs related to its adaption to the high turbidity and eutrophic coastal environment with a low ratio of phosphorus to nitrogen (HNLP). The occurrences of 31 AMGs among 34 cyanophage genomes indicates that AMGs zwf, gnd, speD, petF and those coding for FECH and thioredoxin were more common in coastal areas than in the open ocean and AMGs pebS and ho1 were more prevalent in the open ocean. The occurrence of cyanophage AMGs in different environments might be a reflection of the environmental adaption of their hosts. This study contributes to our understanding of the interactions between cyanobacteria and cyanophages and their environmental adaption to the coastal environment.

Seed weight and number ultimately determine seed yield. Arabidopsis seed number comprised of silique number and seed number per silique (SNS). Comparing seed development and weight, determinants of seed number remain largely uncharacterized. In this study, taking advantage of 107 available Arabidopsis accessions, genome-wide association analysis (GWAS) was employed to identify the candidate genes regulating SNS. GWAS-based genotype and phenotype association analysis identified 38 most significant SNPs marker sites that were mapped to specific chromosomal positions and allowed us to screen for dozens of candidate genes. One of them (PIN3) was selected for functional validation based on gene expression analysis. It is a positive regulator of Arabidopsis SNS. Although silique length of PIN3 loss of function mutant was not significantly changed, its SNS and seed density (SD) were significantly reduced as compared with the wild type. Notably, PIN3 overexpression lines driven by a placenta-specific promoter STK exhibited significantly shorter siliques, slightly reduced SNS, but significant increased SD compared with wild type, suggesting that PIN3 positively regulates SD through inducing ovule primordia initiation regardless of the placenta size. Ovule initiation determines the maximal possibility of SNS, and new genes and mechanism regulating SNS through modulating ovule initiation is worth further investigated.

The main determinant of crop yield is seed yield. In Arabidopsi, many of the genes that regulate seed number are involved in gametophyte and zygote development, and their overexpression cannot increase seed number. However, several mutants with enhanced brassinosteroid (BR) and cytokinin (CK) signals show increased seed number per silique (SNS). Here, we studied the crosstalk between BR and CK signals in regulating SNS through crossing BR- and CK-related mutants. Compared with the mutants ckx3 ckx5 and bzr1-1D , the triple mutant ckx3 ckx5 bzr1-1D with enhanced BR and CK signals formed elongated placentae and showed increased ovule initiation and SNS, indicating that BR and CK synergistically up-regulate SNS. Although BR enhanced the CK signal and CK enhanced the BR signal, while CK partially rescued the phenotypes of BR deficient- and -insensitive mutants, BR could not rescue the phenotypes of CK-deficient mutants. This suggested that CK functions downstream of BR in regulating SNS and that BR regulates SNS through CK-mediated or CK-independent pathways. Comparative transcriptome analyses of ckx3 ckx5 , bzr1-1D , and ckx3 ckx5 bzr1-1D identified known and new genes involved in ovule initiation. The BR-induced transcription factor BZR1 was found to directly interact with the B-type regulator of CK, ARR1, and this interaction enhanced the ability of ARR1 to target and induce genes related to ovule development. Our results demonstrate that BR and CK synergistically increase SNS through co-regulating downstream genes involved in ovule identity and initiation. The genes involved in this process have potential applications in enhancing crop yield.

Strawberry vein banding virus (SVBV)-infected strawberry cells contain cytoplasmic inclusions with isometric particles. To identify the components of the inclusions, green fluorescent protein (GFP) was fused to the carboxy-terminus (C-terminus) of SVBV open reading frames, these constructs were separately transformed into Agrobacterium tumefaciens and infiltrated into Nicotiana benthamiana leaves. Results showed that the SVBV P6 protein assembled into prominent and amorphous inclusion bodies (IBs). To investigate P6 subcellular localization, P6-GFP was ectopically expressed in N. benthamiana leaves by agroinfiltration and then stained with 4’,6-diamidino-2-phenylindole (DAPI). We found the P6 protein accumulated in the nuclei and also formed cytoplasmic IBs with different sizes. To further determine the location of P6 IBs in the cytoplasm, and explore whether the P6 IBs move freely or depend on cytoskeleton and endoplasmic reticulum (ER), the microfilament marker protein (GFP-ABD2-GFP), microtubules marker protein (mCherry-MAP65-1) and ER marker protein (mCherry-HDEL) were separately coexpressed with P6-GFP and into N. benthamiana leaves by agroinfiltration, exhibiting that P6 IBs aligned with cytoskeleton and endoplasmic reticulum. Meanwhile, coinfiltration of P1 and P6 indicated the P6 colocalized with the P1 protein at periphery of cells. The P6 protein contains one C-terminal nuclear localization signal (NLS) region, a P6 protein mutant with a deleted NLS did not localize in the nucleus, did not form IBs, and was unable to facilitate exogenous GFP expression. These results demonstrate that the deleted NLS region is an important P6 domain required for biological functions. In summary, the mobile P6 IBs are associated with ER, microfilaments and microtubules and move along microfilaments to the SVBV P1 protein in the PD.

Although Alteromonas is ubiquitous in the marine environment, very little is known about Alteromonas phages, with only ten, thus far, being isolated and reported on. In this study, a novel double-stranded DNA phage, Alteromonas phage P24, which infects Alteromonas macleodii, was isolated from the coastal waters off Qingdao. Alteromonas phage P24 has a siphoviral morphology, with an icosahedral head, 61 ± 1 nm in diameter, and a tail length of 105 ± 1 nm. Alteromonas phage P24 contains lipids. It has an optimal temperature and pH for growth of 20℃ and 5–7, respectively. A one-step growth curve shows a latent period of 55 min, a rise period of 65 min, and an average burst size of approximately 147 virions per cell. Alteromonas phage P24 has the genome of 46,945 bp with 43.80% GC content and 74 open reading frames (ORFs) without tRNA. The results of the phylogenetic tree, based on the mcp and terL genes, show that Alteromonas phage P24 is closely related to Aeromonas phage phiARM81ld. Meanwhile, phylogenetic analysis based on the whole genome of P24 indicates that it forms a unique viral sub-cluster within Siphoviridae. This study contributes to the understanding of the genomic characteristics and the virus-host interactions of Alteromonas phages.

Background Neonatal hypoxic-ischemic brain damage (HIBD), a leading cause of neonatal mortality, has intractable sequela such as epilepsy that seriously affected the life quality of HIBD survivors. We have previously shown that ion channel dysfunction in the central nervous system played an important role in the process of HIBD-induced epilepsy. Therefore, we continued to validate the underlying mechanisms of TRPV1 as a potential target for epilepsy. Methods Neonatal hypoxic ischemia and oxygen-glucose deprivation (OGD) were used to simulate HIBD in vivo and in vitro. Primarily cultured astrocytes were used to assess the expression of TRPV1, glial fibrillary acidic protein (GFAP), cytoskeletal rearrangement, and inflammatory cytokines by using Western blot, q-PCR, and immunofluorescence. Furthermore, brain electrical activity in freely moving mice was recorded by electroencephalography (EEG). TRPV1 current and neuronal excitability were detected by whole-cell patch clamp. Results Astrocytic TRPV1 translocated to the membrane after OGD. Mechanistically, astrocytic TRPV1 activation increased the inflow of Ca2+, which promoted G-actin polymerized to F-actin, thus promoted astrocyte migration after OGD. Moreover, astrocytic TRPV1 deficiency decreased the production and release of pro-inflammatory cytokines (TNF, IL-6, IL-1β, and iNOS) after OGD. It could also dramatically attenuate neuronal excitability after OGD and brain electrical activity in HIBD mice. Behavioral testing for seizures after HIBD revealed that TRPV1 knockout mice demonstrated prolonged onset latency, shortened duration, and decreased seizure severity when compared with wild-type mice. Conclusions Collectively, TRPV1 promoted astrocyte migration thus helped the infiltration of pro-inflammatory cytokines (TNF, IL-1β, IL-6, and iNOS) from astrocytes into the vicinity of neurons to promote epilepsy. Our study provides a strong rationale for astrocytic TRPV1 to be a therapeutic target for anti-epileptogenesis after HIBD.

p.p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 8.0px Helvetica; color: #2f2a2b} span.s1 {font: 5.5px Helvetica} Na1/H1 exchanger (NHE1) activation is required for multiple microglial functions. We investigated effects of selective deletion of microglial Nhe1 in Cx3cr1-CreER;Nhe1f/f mice on neuroinflammation and tissue repair after ischemic stroke. Infarct volume was similar in corn oil or tamoxifen (Tam)- treated mice at 48 hr and 14 days post-stroke. However, the Tam-treated mice showed significantly higher survival rate and faster neurological function recovery during day 1–14 post-stroke. Deletion of microglial Nhe1 prevented the elevation of CD11b1/CD45low-med microglia in the ischemic hemisphere at day 3 post-stroke, but stimulated expression of Ym1, CD68, TGF-b, IL-10, decreased expression of CD86 and IL-1b, and reduced GFAP1 reactive astrocytes. Moreover, at day 14 post-stroke, enhanced white matter myelination was detected in the microglial Nhe1 deleted mice. In comparison, neuronal Nhe1-null mice (the CamKII-Cre1/2;Nhe1f/f mice) showed a significant reduction in both acute and subacute infarct volume, along with increased survival rate and moderate neurological function recovery. However, these neuronal Nhe1-null mice did not exhibit reduced activation of CD11b1/CD45low-med microglia or CD11b1/CD45hi macrophages in the ischemic brains, and they exhibited no reductions in white matter lesions. Taken together, this study demonstrated that deletion of microglial and neuronal Nhe1 had differential effects on ischemic brain damage. Microglial NHE1 is involved in pro-inflammatory responses during post-stroke brain tissue repair. In contrast, neuronal NHE1 activation is directly associated with the acute ischemic neuronal injury but not inflammation. Our study reveals that NHE1 protein is a potential therapeutic target critical for differential regulation of ischemic neuronal injury, demyelination and tissue repair.

Caveolin-1 (Cav-1) is a principal structural protein of caveolae. Cav-1 has been implicated in cancer progression, but its precise functional roles in pituitary adenoma cells remain largely unclear. In this study, we evidenced that the level of cav-1 was elevated in the invasive pituitary adenoma. Cav-1 knockdown restrained the migration and invasion of pituitary adenoma cells. In cav-1-depleting cells, the expression of miR-145, miR-124 and miR-183 were up-regulated. Further investigation showed that cav-1 knockdown inhibited the nuclear translocation of EGR1, reducing the interaction between EGR1 and KLF5. The resulting free KLF5 promoted the expression of miR-145, miR-124 and miR-183 by binding to their promoters, which was blocked by EGR1. Luciferase reporter assay indicated that miR-145 targeted FSCN1, miR-124 targeted PTTG1IP, and miR-183 targeted EZR in pituitary adenoma cells, respectively. Knockdown of FSCN1, PTTG1IP or EZR suppressed the migration and invasion of pituitary adenoma cells. In conclusion, our data suggested that the elevated cav-1 promoted pituitary adenoma cells migration and invasion by regulating the interaction between EGR1 and KLF5.

In yeast target of rapamycin complex 1 (TORC1) and Tap42-associated phosphatases regulate expression of genes involved in nitrogen limitation response and the nitrogen discrimination pathway. However, it remains unclear whether TORC1 and the phosphatases are required for sensing nitrogen conditions. Utilizing temperature sensitive mutants of tor2 and tap42, we examined the role of TORC1 and Tap42 in nuclear entry of Gln3, a key transcription factor in yeast nitrogen metabolism, in response to changes in nitrogen conditions. Our data show that TORC1 is essential for Gln3 nuclear entry upon nitrogen limitation and downshift in nitrogen quality. However, Tap42-associated phosphatases are required only under nitrogen limitation condition. In cells grown in poor nitrogen medium, the nitrogen permease reactivator kinase (Npr1) inhibits TORC1 activity and alters its association with Tap42, rendering Tap42-associated phosphatases unresponsive to nitrogen limitation. These findings demonstrate a direct role for TORC1 and Tap42-associated phosphatases in sensing nitrogen conditions and unveil an Npr1-dependent mechanism that controls TORC1 and the phosphatases in response to changes in nitrogen quality.

Background Triple-negative breast cancer (TNBC) is an aggressive and deadly breast cancer subtype with limited treatment options. It is necessary to seek complementary strategies for TNBC management. Taraxacum mongolicum , commonly named as dandelion, is a herb medicine with anti-cancer activity and has been utilized to treat mammary abscess, hyperplasia of mammary glands from ancient time in China, but the scientific evidence and action mechanisms still need to be studied. Aim of the study This study was intended to investigate the therapeutic effect and molecular mechanisms of dandelion extract in TNBC cell line. Methodology Dandelion extract was prepared and purified, and then its chemical composition was determined. Cell viability was evaluated by MTT assay. Analysis of cell apoptosis and cell cycle was assessed by flow cytometry. The expression levels of mRNA and proteins were determined by real-time PCR and Western blotting, respectively. Caspase inhibitor Z-VAD-FMK and CHOP siRNA were used to confirm the cell apoptosis induced by dandelion extract. Results Dandelion extract significantly decreased MDA-MB-231cell viability, triggered G2/M phase arrest and cell apoptosis. Concurrently, it caused a markedly increase of cleaved caspase-3 and PARP proteins. Caspase inhibitor Z-VAD-FMK abolished the apoptosis triggered by dandelion extract. The three ER stress-related signals were strongly induced after dandelion treatment, including increased mRNA expressions of ATF4, ATF6, XBP1s, GRP78 and CHOP genes, elevated protein levels of phosphorylated PERK, eIF-2α, IRE1, as well as the downstream molecules of CHOP and GRP78. MDA-MB-231 cells transfected with CHOP siRNA significantly reduced apoptosis induced by dandelion extract. The underlying mechanisms at least partially ascribe to the strong activation of PERK/p-eIF2α/ATF4/CHOP axis. Conclusion ER stress related cell apoptosis accounted for the anti-cancer effect of dandelion extract, and these findings support dandelion extract might be a potential therapeutic approach to treat TNBC.

Tyrosine kinase inhibitors (TKIs) are an effective treatment strategy for non-small cell lung cancer (NSCLC) patients harboring mutations that result in constitutive activation of the epidermal growth factor receptor (EGFR). However, most patients eventually develop resistance to TKIs. This occurs due to additional EGFR mutations or the activation of bypass signaling pathways. In our previous work, we demonstrated that Marsdenia tenacissima extract (MTE) restored gefitinib sensitivity in resistant NSCLC cells with EGFR T790M or K-ras mutations. However, the potential efficacy of MTE in NSCLC cells with resistance mediated by Axl and c-Met, and the related molecular mechanisms need to be elucidated. In this study we evaluated the ability of MTE to restore erlotinib/gefitinib sensitivity in TKI resistant HCC827/ER cells and xenograft mice models. Our results demonstrate that MTE overcomes erlotinib and gefitinib resistance driven by Axl and c-Met in vitro and in vivo. Combination therapy significantly suppressed EGFR downstream molecules and the c-Met and Axl activated bypass signaling pathways. Moreover, we observed that MTE is more efficient at restoring resistance to erlotinib than gefitinib. As the Axl and c-Met mediated bypass pathways share the same downstream signaling cascade as EGFR, simultaneous targeting of these pathways is a promising strategy to overcome acquired resistance of TKIs. Our results demonstrate that MTE treatment attenuates Axl phosphorylation and the associated epithelial-mesenchymal transition, suggesting MTE treatment may be a potential therapeutic strategy for overcoming erlotinib and gefitinib cross-resistance in NSCLC, especially for erlotinib resistance.

Exopolysaccharide has the activity of promoting plants growth. Herein, several agro-industrial residues were used for EPS biosynthesis through solid-state fermentation (SSF) using an EPS producing stain Kosakonia cowanii LT-1. Then the influences of cane molasses powder (CMP), NaNO3 and temperature on EPS biosynthesis through SSF were studied using response surface methodology. Maximum EPS yield (41.62 mg/gds) was obtained in a mixed substrate composed of cane bagasse and broadbean seed capsule (2:1, w/w) supplemented with CMP (5.09%, w/w) and NaNO3 (4.16‰, w/w), which achieved about 30.43% cost savings for substrates. Transcriptomic analysis explained the mechanism of NaNO3 promoting EPS production. Repeated batch SSF was performed eight times with 10% substrate as the seeds of the next SSF cycle successfully. Therefore, the fermented SSF substrate containing K. cowanii LT-1 and its EPS was applied in maize growth, which could promote seed germination rate and growth vigor of maize. Hence, fermented SSF substrates would be an ideal candidate for application as a plant growth promoter in agriculture field.

Brassinosteroid (BR) is a family of bioactive steroid hormones that plays vital roles in plant growth and development. The BR-mediated regulation of plant growth and architecture has been well studied. However, relatively few studies have investigated the BR-related regulation of reproductive development because of the difficulties in excluding non-specific regulation and secondary responses from severe vegetative phenotypes and poor nutritional status. Furthermore, differentially regulating the BR signal in vegetative and reproductive organs is problematic. Thus, establishing a method for modulating the BR signal only in reproductive organs or during reproductive developmental stages will be beneficial. Additionally, the utility of BR applications for crop production is limited because of deleterious side-effects, including the associated decrease in the planting density and lodging resistance. Moreover, enhancing the BR signal may lead to feedback inhibition. In this study, we developed a transformation system for modulating the BR signal differentially during reproductive and vegetative developmental stages. This system involves transformations with different combinations of a reproductive tissue-specific promoter, coding sequences that increase or decrease the BR signal, and various genotypic backgrounds with enhanced or decreased BR signals. The enhanced BR signal generated in transformants was targeted to reproductive organs without affecting vegetative organs. This system may be useful for studying the BR-specific regulation of plant reproductive development and shows promise for optimizing seed yield.

Glioblastoma is a common malignant primary intracranial tumor characterized by rapid invasive growth and a high recurrence rate after surgery. MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis, and abnormal miRNA expression is associated with the occurrence and progression of various tumors, including glioblastomas. The aim of this study was to determine the levels of miR-148a and integrin subunit alpha 9 (ITGA9) in glioblastoma tissues and cells and their involvement in cancer cell proliferation and migration. Glioblastoma tissues from 19 patients and two glioblastoma cell lines (U87 and LN229) were used in this study. The effects of miR-148a on cell viability, proliferation, colony formation, migration, and invasion were assessed. Glioblastomas were xenografted in nude mice to examine the effects of miR-148a overexpression on tumor growth in vivo. Levels of ITGA9 mRNA and protein in glioblastoma tissues were detected by quantitative reverse transcription PCR and western blot analysis, respectively. The interaction between miR-148a and ITGA9 was determined by a dual-luciferase reporter gene assay. We found that the overexpression of miR-148a decreases the proliferation, clustering, migration, and invasiveness of U87 and LN229 cells and inhibits the tumorigenicity of xenografted glioblastomas. We confirmed that ITGA9 is the target of miR-148a. Restoration of ITGA9 expression reversed the decreased viability, migration, and invasiveness of glioblastoma cells induced by miR-148a overexpression. Our findings indicate that miR-148a can suppress the malignant phenotype of glioblastoma by targeting ITGA9 and identify ITGA9 as a potential therapeutic target for glioblastoma.

Background Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1β expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. Methods Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1β, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. Results Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1β, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1β by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1β release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. Conclusions TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1β mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h). Electronic supplementary material The online version of this article (10.1186/s12974-019-1487-3) contains supplementary material, which is available to authorized users.

Persistent endoplasmic reticulum (ER) stress in neurons is associated with activation of inflammatory cells and subsequent neuroinflammation following traumatic brain injury (TBI); however, the underlying mechanism remains elusive. We found that induction of neuronal-ER stress, which was mostly characterized by an increase in phosphorylation of a protein kinase R-like ER kinase (PERK) leads to release of excess interferon (IFN)β due to atypical activation of the neuronal-STING signaling pathway. IFNβ enforced activation and polarization of the primary microglial cells to inflammatory M1 phenotype with the secretion of a proinflammatory chemokine CXCL10 due to activation of STAT1 signaling. The secreted CXCL10, in turn, stimulated the T-cell infiltration by serving as the ligand and chemoattractant for CXCR3+T-helper 1 (Th1) cells. The activation of microglial cells and infiltration of Th1 cells resulted in white matter injury, characterized by impaired myelin basic protein and neurofilament NF200, the reduced thickness of corpus callosum and external capsule, and decline of mature oligodendrocytes and oligodendrocyte precursor cells. Intranasal delivery of CXCL10 siRNA blocked Th1 infiltration but did not fully rescue microglial activation and white matter injury after TBI. However, impeding PERK-phosphorylation through the administration of GSK2656157 abrogated neuronal induction of IFNβ, switched microglial polarization to M2 phenotype, prevented Th1 infiltration, and increased Th2 and Treg levels. These events ultimately attenuated the white matter injury and improved anxiety and depressive-like behavior following TBI.SIGNIFICANCE STATEMENTA recent clinical study showed that human brain trauma patients had enhanced expression of type-1 IFN; suggests that type-1 IFN signaling may potentially influence clinical outcome in TBI patients. However, it was not understood how TBI leads to an increase in IFNβ and whether induction of IFNβ has any influence on neuroinflammation, which is the primary reason for morbidity and mortality in TBI. Our study suggests that induction of PERK phosphorylation, a characteristic feature of ER stress is responsible for an increase in neuronal IFNβ, which, in turn, activates microglial cells and subsequently manifests the infiltration of T cells to induce neuroinflammation and subsequently white matter injury. Blocking PERK phosphorylation using GSK2656157 (or PERK knockdown) the whole cascade of neuroinflammation was attenuated and improved cognitive function after TBI.

The management of triple-negative breast cancer (TNBC) is challenging due to the aggressive behavior, lack of therapeutic options and relatively poor prognosis. Xihuang pill (XHP) is a well-known Traditional Chinese Medicine with anticancer activity. The aim of the present study was to investigate whether the aqueous extract of XHP (AEXHP) has anti-proliferative activity against the Hs578T TNBC cell line, and to elucidate its molecular mechanisms of action. First, an MTT assay was used to evaluate the anti-proliferative activity of AEXHP on the Hs578T cell line; furthermore, the cell cycle distribution, mitochondrial membrane potential and apoptotic rate were determined by flow cytometry, and western blot analysis was used to assess the expression of apoptosis and cell cycle regulatory proteins to investigate the mechanisms of action. The results revealed that the cell viability was significantly inhibited by AEXHP in a dose- and time-dependent manner. Apoptosis and mitochondrial membrane potential loss were detected, and after treatment with 4, 8 and 12 mg/ml AEXHP for 24 h, cleaved caspase-3 was 1.70-, 1.81- and 1.84-fold of that of the control, while procaspase-3, procaspase-8, cleaved caspase-8, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax) and the Bcl-2/Bax ratio were not significantly affected. Cell cycle analysis revealed that treatment with AEXHP led to S-phase arrest of Hs578T cells. Furthermore, AEXHP treatment resulted in decreased expression of cyclin A and cyclin dependent kinase 2 (CDK2), and increased expression of cyclin E and p21Cip1, as compared to the control group. In conclusion, the viability of Hs578T cells was significantly inhibited by AEXHP in a dose- and time-dependent manner, the likely mechanisms of which being induction of apoptosis, probably via the intrinsic, Bcl-2-independent pathway, and cell cycle arrest in S phase due to decreased expression of cyclin A and CDK2, and increased expression of cyclin E and p21Cip1.

Evidence indicates that inflammation plays a crucial role in depression. Therefore, new antidepressants might be identified by screening drugs for their anti-inflammatory actions. Sertraline hydrochloride (SERT), a widely used antidepressant, has anti-inflammatory effects in clinical studies, but the mechanism involved is unclear. In this study, we used cell and molecular biology to determine the possible anti-inflammatory mechanism of SERT in vivo and in vitro. Experimental data from the in vivo study showed that mice exposed to chronic unpredictable mild stress (CUMS) had significantly higher levels of major inflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin-1β [IL-1β] and inducible nitric oxide synthase [iNOS]) in peripheral and central tissues compared with the control group. Treatment of CUMS mice with SERT significantly reduced the levels of these inflammatory cytokines and inhibited the phosphorylation of nuclear factor-κB (NF-κB) and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha (IκB-α). Moreover, SERT reduced serum levels of transaminase in CUMS mice. Our in vitro study revealed that SERT suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. SERT also inhibited the TNF-α-induced nuclear translocation of NF-κB by inhibiting IκB-α phosphorylation. Furthermore, SERT inhibited TNF-α-induced inflammatory cytokines in BV2 microglia cells. SERT directly bound to TNF-α and TNF-α receptor 1 (TNFR1) to potently block TNF-α/TNFR1-triggered signaling. These results indicate that SERT might treat depression by inhibiting the activation of microglia via the NF-κB signaling pathway. This study provides a basis for the research and development of antidepressants that act to reduce inflammation and the expression of inflammatory mediators.

The management of patients with triple‑negative breast cancer is challenging due to the lack of effective therapeutic options, aggressive behavior and relatively poor prognosis. Xi Huang pills (XHP) are a well‑known traditional Chinese medicine that demonstrate anticancer activities. The aim of the present study was to investigate the antitumor effects of XHP on MDA‑MB‑231 cells in vitro and in vivo, and its potential underlying molecular mechanisms. In the present study, an MTT assay was used to evaluate the antiproliferative activity of XHP on MDA‑MB‑231 cells. In order to investigate the effects further, cell cycle distribution, apoptosis and mitochondrial membrane potential assays were performed, as well as western blot analyses. In addition, a tumor xenograft model was employed to investigate the effects of XHP in vivo. The results of the MTT assay demonstrated that the viability of MDA‑MB‑231 cells was markedly inhibited by XHP in a dose‑ and time‑dependent manner. The inhibitory effect of XHP on the viability of MDA‑MB‑231 cells was greater when compared with MCF‑10A cells. An increase in apoptosis and loss of mitochondrial membrane potential was observed following 4, 8 and 12 mg/ml XHP treatment of MDA‑MB‑231 cells. The protein expression levels of cleaved caspase‑3 were increased by 1.62‑, 2.13‑ and 2.19‑fold, respectively, when compared with the untreated controls, whereas no effects on the expression of B‑cell lymphoma 2 (Bcl‑2) or Bcl‑2‑associated X protein (Bax) were observed. The results of the cell cycle distribution assay analysis demonstrated that XHP treatment arrested cells at the G2/M phase. In addition, XHP treatment decreased the expression of cyclin A and increased the expression of p21Cip1. In vivo experiments revealed that XHP inhibited the growth of MDA‑MB‑231 xenograft tumors without body weight loss, and demonstrated similar effects on the protein expression levels of cleaved caspase 3, cyclin A and p21Cip1 as observed in vitro. In conclusion, the viability of MDA‑MB‑231 cells was inhibited by XHP in a dose‑dependent, time‑dependent and cell‑selective manner in vitro, and the potential underlying mechanisms may involve apoptosis and cell cycle arrest at the G2/M phase. XHP may induce apoptosis in MDA‑MB‑231 cells via the intrinsic pathway, which does not involve the Bcl‑2/Bax ratio. G2/M phase arrest may have been due to the integrated action of decreased cyclin A expression and increased p21Cip1 expression. In addition, XHP inhibited the growth of xenograft tumors in the absence of body weight loss in vivo.

A novel cyanophage, S-B64, which can infect marine Synechococcus WH8102, was isolated from the coastal waters of the Yellow Sea using the liquid serial dilution method. Morphological study by transmission electron microscopy revealed that the cyanophage belongs to Podovirus. It’s genome, which was completely sequenced, contains a 151,867 bp DNA molecule with a G+C content of 41.78% and 186 potential open reading frames. The functions of the genes include cyanophage structure, cyanophage packaging, DNA replication and regulation. After primary characterization, it was found that the latent period is about 3 h, and it lysed after 8 h, the burst size is about 23 virions per cell. This information will provide an important benchmark for further research on the interaction between cyanophages and their hosts.

Viral infections cause plant chlorosis, stunting, necrosis or other symptoms. The down-regulation of chloroplast-related genes (ChRGs) is assumed to be responsible for chlorosis. We identified the differentially expressed genes (DEGs) in Rice stripe virus (RSV)-infected Nicotiana benthamiana, and examined the contribution of 75 down-regulated DEGs to RSV symptoms by silencing them one by one using Tobacco rattle virus (TRV)-induced gene silencing. Silencing of 11 of the 75 down-regulated DEGs caused plant chlorosis, and nine of the 11 were ChRGs. Silencing of a down-regulated DEG encoding the eukaryotic translation initiation factor 4A (eIF4A) caused leaf-twisting and stunting that were visible on RSV-infected N. benthamiana. A region of RSV RNA4 was complementary to part of eIF4A mRNA and virus-derived small interfering (vsiRNAs) from that region were present in infected N. benthamiana. When expressed as artificial microRNAs, those vsiRNAs could target NbeIF4A mRNA for regulation. We provide experimental evidence supporting the association of ChRGs with chlorosis and show that eIF4A is involved in RSV symptom development. This is also the first report demonstrating that siRNA derived directly from a plant virus can target a host gene for regulation.

Gene associated with retinoid‑interferon‑induced mortality 19 (GRIM‑19) and the liver kinase B1 (LKB1) gene, two types of tumor suppressor gene, have been demonstrated to have important roles in breast carcinogenesis. The present study developed a dual expression plasmid that co‑expressed GRIM‑19 and LKB1, and evaluated the combined effects of the two genes against breast cancer in vitro and in vivo. Transfection with a plasmid for the simultaneous expression of GRIM‑19 and LKB1 (pGRIM19‑LKB1) into MCF‑7 breast cancer cells significantly inhibited the proliferation, colony formation, migration and invasion compared with the effects of transfection with either pGRIM‑19 or pLKB1 alone. Furthermore, transfection with pGRIM19‑LKB1 induced enhanced levels of apoptosis and cell cycle arrest at G0/G1 stage in MCF7 cells compared to the effects of pGRIM‑19 or pLKB1 alone. An in vivo experiment using an MCF‑7 xenograft tumor model demonstrated that intravenous injection of pGRIM19‑LKB1 had an enhanced effect on tumor growth inhibition compared to that of pGRIM‑19 or pLKB1 alone. In conclusion the findings of the present study suggested that transfection with eukaryotic plasmid for the simultaneous expression of GRIM‑19 and LKB1 more effectively suppressed the growth of breast cancer in vitro and in vivo, and may therefore have therapeutic potential for the treatment of human breast cancer.

The present study was designed to evaluate the antioxidant activities of anthocyanins obtained from purple sweet potato (PSPAs) and their protective effect on hepatic fibrosis induced by carbon tetrachloride (CCl4) administration in mice. Anthocyanins are natural food colorants. PSPAs showed high scavenging effects against 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH) and . Tested mice were orally treated with PSPAs every day for three weeks and in combination with CCl4 at last. The results indicated that treatment with CCl4 caused hepatotoxicity which was assessed by an increase in the levels of relative liver weight, serum aspartate aminotransferase and alanine aminotransferase, as well as in hepatic lipid peroxide malondialdehyde. Moreover, the activities of superoxide dismutase and glutathione peroxidase were found reduced. However, these changes were inhibited by the treatment with PSPAs prior to the administration of CCl4. The biochemical alternations were accompanied by histopathological cha...

Highlights • A novel RT-LAMP method was developed to detect canine distemper virus (CDV). • A set of four primers were designed to target the H gene for the specific detection of wild-type CDV variants. • The assay was 100-fold more sensitive than conventional RT-PCR. • The system showed a preference for wild-type CDV, and exhibited less sensitivity to CPV., Although widespread vaccination against canine distemper virus (CDV) has been conducted for many decades, several canine distemper outbreaks in vaccinated animals have been reported frequently. In order to detect and differentiate the wild-type and vaccine strains of the CDV from the vaccinated animals, a novel reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was developed. A set of four primers—two internal and two external—were designed to target the H gene for the specific detection of wild-type CDV variants. The CDV-H RT-LAMP assay rapidly amplified the target gene, within 60 min, using a water bath held at a constant temperature of 65 °C. The assay was 100-fold more sensitive than conventional RT-PCR, with a detection limit of 10−1 TCID50 ml−1. The system showed a preference for wild-type CDV, and exhibited less sensitivity to canine parvovirus, canine adenovirus type 1 and type 2, canine coronavirus, and canine parainfluenza virus. The assay was validated using 102 clinical samples obtained from vaccinated dog farms, and the results were comparable to a multiplex nested RT-PCR assay. The specific CDV-H RT-LAMP assay provides a simple, rapid, and sensitive tool for the detection of canines infected with wild-type CDV from canines vaccinated with attenuated vaccine.

Gene associated with retinoid-interferon (IFN)-induced mortality 19 (GRIM-19), a novel IFN-β/retinoic acid-inducible gene product, has been identified as a potential tumor suppressor, which is associated with the inhibition of tumor growth. GRIM-19 has been demonstrated to be downregulated in the ovarian tissue of patients with breast cancer, however, its role in breast cancer remains to be fully elucidated. In the present study, a recombinant eukaryotic expression plasmid carrying GRIM-19 was constructed and then transfected into the MCF7 human breast cancer cell line to examine its effects on breast cancer cell growth, migration and invasion using several in vitro approaches. The results demonstrated that upregulation GRIM-19 in the MCF7 cells significantly inhibited cell proliferation, colony formation, migration and invasion, and induced cell apoptosis. Additionally, upregulation of GRIM-19 also suppressed the secretion of urokinase-type plasminogen activator (u-PA), matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF). It was also demonstrated that the activation of signal transducer and activator of transcription 3 (STAT3) was downregulated by the expression of GRIM-19. These results revealed that overexpression of the GRIM-19 gene may be an effective approach to control the growth and invasion of human breast cancer cells.

RNA silencing is a common mechanism that plays a key role in antiviral defense. To overcome host defense responses, plant viruses encode silencing-suppressor proteins to target one or several key steps in the silencing machinery. Here, we report that the P6 protein encoded by Strawberry vein banding virus (SVBV) is an RNA silencing suppressor through Agrobacterium-mediated co-infiltration assays. SVBV P6 protein can suppress green fluorescent protein (GFP) gene silencing induced by single-stranded RNA but not by double-stranded RNA. The P6 protein can also inhibit systemic silencing of GFP through interfering the systemic spread of GFP silencing signal. Subcellular localization study indicated that P6 protein formed irregular bodies and distributed in both cytoplasm and nucleus of Nicotiana benthamiana cells. Furthermore, deletion analysis indicated that a nuclear localization signal (NLS, aa 402-426) in the P6 protein is responsible for the silencing suppression efficiency. In addition, expression of the P6 protein via a Potato virus X (PVX)-based vectors induced more severe mosaic symptoms in N. benthamiana leaves, and transgenic N. benthamiana plants expressing P6 showed obvious vein yellowing as well as severe mosaic symptoms in leaves. Taken together, our results demonstrates that SVBV P6 is a suppressor of RNA silencing, possibly acting at a upstream step for dsRNA generation.

Cellular redox status and oxygen availability influence the product formation. Herein, decreasing agitation speed or adding vitamin C (Vc) achieved the 2,3-BDL yield of 0.40 g g(-1) or 0.39 g g(-1)glucose under batch fermentation, respectively. To our knowledge, this is the highest 2,3-BDL yield reported so far for Paenibacillus polymyxa without adding acetic acid. The NADH/NAD(+) ratio and 2,3-BDL titer could be increased significantly by reducing the agitation speed or adding Vc, indicating that the enhancement of 2,3-BDL is closely associated with the adjustment of NADH/NAD(+) ratio. Especially, Vc addition elevated the 2,3-BDL titer from 43.66 g L(-1) to 71.71 g L(-1) within 54 h under fed-batch fermentation. This is the highest titer of 2,3-BDL so far reported for P. polymyxa from glucose fermentation. This work provides a new strategy to improve 2,3-BDL production and helps us to understand the responses of P. polymyxa to extracellular oxidoreduction potential.

This study examines the bioleaching of municipal solid waste incineration fly ash by Aspergillus niger, and its effect on the fungal morphology, the fate of the ash particles, and the precipitation of metallic salt crystals during bioleaching. The fungal morphology was significantly affected during one-step and two-step bioleaching; scanning electron microscopy revealed that bioleaching caused distortion of the fungal hyphae (with up to 10μm hyphae diameter) and a swollen pellet structure. In the absence of the fly ash, the fungi showed a linear structure (with 2–4μm hyphae diameter). Energy-dispersive X-ray spectroscopy and X-ray diffraction confirmed the precipitation of calcium oxalate hydrate crystals at the surface of hyphae in both one-step and two-step bioleaching. Calcium oxalate precipitation affects bioleaching via the weakening of the fly ash, thus facilitating the release of other tightly bound metals in the matrix.